method of fabricating a membrane having a tapered pore

ABSTRACT

The present invention relates to a method of fabricating a membrane having a tapered pore, to a polymeric membrane having a tapered pore and to uses of such polymeric membrane.

The present invention relates to a method of fabricating a membranehaving a tapered pore, to a polymeric membrane having a tapered pore andto uses of such polymeric membrane.

Suspended bilayers are usually formed across a solid support thatcontains an aperture/pore. It is known that the area of theaperture/pore across which the bilayer spans, influences the stabilityof the bilayer. On the one hand, the smaller the area is, the longer thebilayer generally stays stable. On the other hand, the smaller the areaof the bilayer is, the longer it takes to introduce a pore protein intothe suspended part of the formed bilayer.

In order to form the bilayer, the two sides of the membranes are usuallybrought into contact with liquids, which contain the components of thebilayer. While the liquid is applied on one side, the liquid on theother side of the membrane is usually flowed across the aperture/pore.This procedure leads to the spontaneous formation of a lipid bilayeracross the aperture within a single trial or after several trials.

A number of research groups have studied suitable solid supports forlipid bilayer formation.

1) Dogan et al. claimed a silicon/silicon oxide support or a polyimidesupport that contains an circular aperture with diameters in the rangebetween 1 μm and 50 μm. The aspect ratio between aperture diameter andmembrane thickness as well as the surface treatment was not furtherspecified in this application.

2) Takeuchi et al. taught in a quite general way a tapered aperture thatnarrows continuously towards the aperture edge. Their material of choiceis Si/SiO₂. The geometrical dimensions of the aperture and of themembranes were not specified in the claims. Details about thegeometrical dimensions of the continuous taper and the surface treatmentwere not further specified in this application.

3) Pohl et al. reported about a lipid bilayer forming technique thatutilises an aperture that is made in glass or in plastic. The diameterrange they claim was 50 μm to 300 μm. No further details about aspectratio or surface treatment were given.

4) Heath et al. filed a patent application in 2004, which describes themembrane with an apertures with diameters between 1 μm and 200 μm madefrom silicon wafers. They suggest that the surface of the membrane iscovered with an insulating material like SiO2 or SixNy. In addition tothis coverage, surface modification by silanisation is introduced inorder to increase the adhesion of biological material to the membranesurface. Specific details to the shape of the aperture and the aspectratio between aperture diameter and thickness of the membrane close tothe aperture were not given.

5) Ogier et al. have published two papers in 2000 and 2001,respectively, describing the fabrication of apertures in SU8photoresist-based membranes that were covered with an additional goldlayer that could be modified by using thiol chemistry. The thickness ofthe central membrane was 10 μm, the aperture diameter was about 100 μm.Further considerations about the aspect ratio, especially for the caseof aperture diameters smaller than 100 μm were not mentioned.

In order to support bilayers to build so-called suspended bilayers forpractical device applications, solid structures with apertures arerequired. These usually have the shape of thin membranes that have asingle or multiple openings (apertures). The membrane thickness as wellas the diameter of the aperture, and the surface properties are believedto represent the major factors that influence the dynamics of formingbilayer membranes across the apertures (FIG. 1).

The aperture area influences the long-term stability of a bilayer: Ingeneral, bilayers across small area aperture are more resilient thanbilayers that cover large areas.

The aspect ratio—defined as the ratio between the diameter of theaperture (d) and the membrane thickness (h)—influences the structuralproperties of the interface between the edges of the solid membrane andthe fluid bilayer. The aperture edges should be as sharp as possible—inother words, the thickness of the membrane [at the membrane-to-bilayertransition] should be as small as possible.

This leads to the requirement of very thin membranes at the apertureedges—which need to be still mechanically stable. For example, apertureedges with conical shapes that have sharp angles are a possiblesolution. In contrast to this, apertures with low aspect ratios tend tohave cylindrical, tube-like shapes which tend become blocked with theannulus forming organic phase or to trap air bubbles, preventing bilayerformation.

The surface properties of the membrane and specifically those of theedges of the aperture are important for the bilayer formation as well.Hydrophobic surface properties are preferable in order to support thewetting of the membrane with the hydrophobic core of the lipid bilayer.

Accordingly, it was an object of the present invention to provide for amethod of fabricating a membrane having a tapered pore which enables theformation of high aspect ratios at low aperture diameters. It was alsoan object of the present invention to provide for membranes that arethin and yet stable and which can be handled easily at a reduced risk ofrupture. Moreover, it was an object of the present invention to providefor a cheaper manufacturing method in comparison to silicon-basedmembranes.

It was also an object of the present invention to provide acorresponding membrane having a tapered pore with a diameter in therange between 1 μm and 30 μm.

The objects of the present invention are solved by a method offabricating a membrane having a tapered pore, said method comprising thesteps:

-   -   a) providing a substrate,    -   b) depositing an anti-sticking layer on said substrate,    -   c) depositing a first polymeric layer of a first polymeric        material on said anti-sticking layer,    -   d) introducing a first aperture into said first polymeric layer,        said first aperture having a first diameter,    -   e) depositing a second polymeric layer of a second polymeric        material onto said first polymeric layer, which second polymeric        layer is located on said first polymeric layer and wherein a        part of said second polymeric material also extends into and        fills said first aperture,    -   f) introducing a second aperture into said second polymeric        layer, said second aperture having a second diameter which is        larger than said first diameter, said second aperture being        aligned with said first aperture, such that said part of said        second polymeric material, which extends into and fills said        first aperture, becomes removed,    -   g) repeating steps e) and f) n-times, thus generating a third,        fourth, . . . (n+2)th polymeric layer having a third, fourth, .        . . (n+2)th aperture in said third, fourth, . . . (n+2)th        polymeric layer, said third, fourth, . . . (n+2)th aperture        having a third, fourth, . . . (n+2)th diameter which is        increasingly larger with increasing n, n being an integer from 0        to 10,        said method thus resulting in a polymeric membrane on an        anti-sticking layer and a substrate, said polymeric membrane        having a tapered pore comprising (n+2) apertures of increasing        diameter, said (n+2) apertures being aligned with each other to        form said tapered pore.

In one embodiment, said (n+2) apertures are concentrically aligned.

In one embodiment, said first diameter is in the range from 1 nm to 100μm, preferably from 1 nm to 10 μm.

In one embodiment, said first polymeric layer has a height in the rangefrom 0.3 nm to 20 μm, preferably 0.3 nm to 2 μm, and wherein said secondand, if present, third, fourth, . . . (n+2)th polymeric layer has aheight in the range from 500 nm to 1 mm, preferably 5 μm to 50 μm.

In one embodiment, the ratio of said first diameter: height of saidfirst polymeric layer is ≧3.

Preferably, said substrate is made of a material selected from the groupcomprising glass, silicon, silicon oxide, silicon nitride, GaAs,saphire, polycarbide, polycarbonate.

In one embodiment, said anti-sticking layer is made of a materialselected from the group comprising metals with a weak adhesion to thesubstrate, such as gold, silver, platinum, titanium, aluminum, alloyshereof, fluorosilanes, mica, carbon, water-soluble materials (e.g. CaO,Ca(OH)₂), heat-disposable materials and has a thickness in the rangefrom 10 nm to 100 nm.

In one embodiment, said first, second and, if present, third, fourth, .. . (n+2)th polymeric material is, independently, at each occurrence,selected from the group comprising of resists for optical lithography,electron beam lithography, and imprint lithography.

In one embodiment, step b) occurs by a method selected from evaporation,sputtering, e-gun evaporation, gas-phase-deposition, sublimation,electro-chemical deposition.

In one embodiment, steps c) and e) are independently performed by amethod selected from spin coating, dip coating, spray coating, vacuumdeposition, Langmuir Blodgett techniques, deposition from the gas-phase.

In one embodiment, steps d) and f) are independently performed by amethod selected from optical lithography, electron beam lithography,imprint lithography, and focused ion beam etching.

In one embodiment, in steps d) and f), a plurality of first and secondapertures are introduced.

Preferably, said resists for optical lithography, electron beamlithography and imprint lithography are selected from the groupcomprising negative and positive tone resists for optical lithography,electron beam lithography and imprint lithography.

In one embodiment, the method according to the present invention furthercomprises the step:

-   -   h) removing said substrate by peeling off said polymeric        membrane on and together with said anti-sticking layer from said        substrate.

Preferably, the method according to the present invention furthercomprises the step:

-   -   i) removing said anti-sticking layer by dry etching, such as dry        argon-etching, O₂ plasma etching or wet chemical etching, such        as KI/I₂-etching to remove gold, etching using a strong base to        remove aluminum.

In one embodiment, the method according to the present invention furthercomprises the step:

-   -   i′) introducing an aperture into said anti-sticking layer by dry        etching, such as dry argon-etching, or wet chemical etching,        such as KI/I₂-etching, and using said first polymeric layer as        an etching mask.

In one embodiment, the method according to the present invention furthercomprises the step:

-   -   h′) selectively etching said polymeric membrane on said        anti-sticking layer and said substrate by using dry plasma        etching, such as O₂ plasma etching, or chemical wet etching,        such as oxidation in H₂SO₄/H₂O₂, to reduce the total thickness        of said polymeric membrane, and, optionally, removing said        substrate by peeling off said polymeric membrane on and together        with said anti-sticking layer from said substrate.

In one embodiment, the method according to the present invention furthercomprises the step:

-   -   k) selectively etching said polymeric membrane on said        anti-sticking layer and, if present, on said substrate, by using        dry plasma etching, such as O₂-plasma etching, or chemical wet        etching, such as oxidation in H₂SO₄/H₂O₂ to reduce the total        thickness of said polymeric membrane, and, optionally, removing        said substrate, if present, by peeling off said polymeric        membrane on and together with said anti-sticking layer from said        substrate.

In one embodiment, after removal of said substrate, said polymericmembrane and/or, if present, said anti-sticking layer is/are furthersurface modified by a method selected from

-   -   a) immobilizing functional groups or molecules thereon to match        the surface properties of a lipid bilayer to be introduced into        said first aperture or into said aperture in said anti-sticking        layer,    -   b) surface activation by introducing OH-groups through O₂-plasma        treatment    -   c) enhancing the surface roughness by etching, such as Ar, CHF₃,        CF₄, O₂-plasma and combinations thereof,    -   d) deposition of one or several functional layers, e.g. by dip        coating, gas-phase deposition or evaporation, and any        combination of steps a)-d).

Preferably, said functional layer is selected from fluorosilanes,alkylsilanes, fluorinated plasma components from CHF₃, CF₄.

The objects of the present invention are also solved by a polymericmembrane having a tapered pore and comprising (n+2) polymeric layers asdefined above, wherein said tapered pore is formed by (n+2) apertures ofdiffering diameter within said (n+2) polymeric layers, n being aninteger from 0 to 10, said n+2 apertures being aligned with each otherto form said tapered pore.

Preferably, said (n+2) apertures are concentrically aligned.

In one embodiment, said tapered pore, at its smallest diameter, has aratio of diameter of said pore to height of said polymeric layer at saidsmallest diameter of said pore of ≧3.

In one embodiment, the polymeric membrane is fabricated by the methodaccording to the present invention.

In one embodiment, the polymeric membrane according to the presentinvention has a plurality of tapered pores, as defined above.

In one embodiment, the polymeric membrane according to the presentinvention further comprises a lipid bilayer spanning the pore at itssmallest diameter and, optionally, having biological membrane proteins,such as pore proteins or channel proteins incorporated in said lipidbilayer.

The objects of the present invention are also solved by the use of amembrane according to the present invention for forming a lipid bilayer.

The objects of the present invention are also solved by the use of apolymeric membrane according to the present invention for physiologicalmeasurements, such as patch clamp measurements or as an electronicsensor.

The term “anti-sticking layer”, as used herein, is meant to describe alayer which, because of its low adhesion to a substrate, allows thepeeling off of said anti-sticking layer, together with any additionallayer on top of it, from said substrate. It is clear from the foregoingthat said anti-sticking layer should only weakly adhere to the flatsurface of a substrate, but it should have better adhesion properties toa layer attached to the anti-sticking layer on the other side.

The exact choice of material of the anti-sticking layer thereforedepends on the material of the substrate and can be determined bysomeone skilled in the art using his knowledge without undueexperimentation. For example, someone skilled in the art knows that alayer of gold has very little adhesion to a silicon oxide surface. Ingeneral, the anti-sticking layer is made of a material selected from thegroup comprising metals, oxides, plastics or other organic componentswhich show a weak adhesion to the material of which the substrate may bemade. Useful examples of materials for the anti-sticking layer are gold,silver, platinum, titanium, mica, carbon, fluorosilanes, water-soluberesists, and heat-disposable thin-film materials. The term “pore” asused herein is meant to refer to an opening in a membrane which is madeof a plurality, i.e. at least 2, apertures on top of each other, whereineach aperture is formed in or by a layer within the membrane. A membranein accordance with the present invention has at least 2 layers. In oneembodiment, the membrane has (n+2) layers, wherein n is an integerselected from 0 to 10, i.e. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

The term “aspect ratio”, as used herein, is defined as the ratio betweenthe diameter (d) of the aperture and the height/thickness (h) of thelayer/membrane at that particular diameter (d:h). Preferably, in atapered pore according to the present invention, the term “aspect ratio”is meant to refer to the ratio of diameter to height at the smallestdiameter, i.e. diameter of the smallest aperture to height of the layerforming this aperture. The height at the smallest diameter is the heightof the layer forming the aperture with the smallest diameter.

In the following we present a solution for realising mechanically stablemembranes with apertures of a diameter of the magnitude of 10 μm or evenbelow and large aspect ratios. As described above, we suggest that theaspect ratio (ratio between the diameter of the aperture/pore (d) andthe thickness (h) of the membrane that holds the aperture) is importantfor the bilayer formation dynamics. Aspect ratios of 3:1 and higher arepreferable, while d should be equal or smaller than 10 μm and h smallerthan 2 μm.

The basic principle of the design is shown in FIG. 1.

The optimisation of the aspect ratio requires stable sub-10 μm thickmembranes that contain a single aperture or an array of apertures, eachof them having a diameter of preferably 10 μm or smaller. Furthermore,the fabrication process should be as cheap as possible and thereforerely on polymeric materials or hybrid structures of polymeric materialsand inorganic materials such as oxides (silicon oxides, siliconnitrides, aluminium oxides).

The fabrication approach in order to realise free-standing and stablesub-10 μm thick polymeric membranes on wafer-scale and at low costs isdescribed in the following:

A thin membrane layer 1 is deposited by a deposition method on asacrificial solid support and a single aperture is introduced byapplying a patterning method. The diameter of the aperture is in therange 1 nm to 100 μm, preferably smaller than 10 μm. On top of thisfirst layer, a second membrane layer 2 (“supporting layer”) isintroduced by a deposition method and a second aperture is introduced bya patterning method. The diameter of the second aperture is preferablylarger than the diameter of the first aperture. The thickness of layer 1is in the range 0.3 nm to 20 μm, preferably in the range 0.3 nm to 2 μm.The thickness of supporting layer 2 is in the range between 500 nm and 1mm, preferably in the range 5 μm to 50 μm.

The aperture of the layer 1 and that of layer 2 shall be aligned in away that the smaller aperture is partly or completely open (FIG. 1). Asused herein, such a structure is herein also referred to as a taperedpore. This two-layer structure can be extended by one ore moreadditional layers, each of them containing an aperture (FIG. 2). Aschematic view of the approach is shown in FIG. 3 and will be explainedin detail in the example section.

It is also possible to include more than one aperture or pore into layer1 and the following layers, in order to build up pore arrays. The numberof array elements could vary between 2 and more than 100.

The patterning method can be selected from a number of differentapproaches, such as optical lithography, electron beam lithography,imprint lithography, and focused ion beam etching.

The deposition method can be selected from a number of differentapproaches: spin-coating, vacuum deposition, spray coating, dip-coating.

The materials that are potentially useful for layer 1 and layer 2 (andthe following layers) are polymers that can be deposited and patternedby the mentioned deposition and patterning methods, respectively.Preferred polymers, useful in accordance with the present invention, arepositive and negative tone resists. Example polymers are negative-toneSU8® resists, positive and negative-tone AZ®-resists, PMMA electron beamresists, UV6 mix & match® resists.

Extension of Aperture Through Back-layer

In a variation of the present inventors' approach, the underlying “layer0” in FIG. 3 (e.g., gold) can be used as an anchor layer for theimmobilisation of specific molecules, which perform a certainfunctionality, e.g. to match the surface properties to the properties ofthe bi-layer. As shown in FIG. 4, the “layer 0” could be selectivelyetched by using dry plasma or wet-etching and by using the polymer“layer 1” as etching mask.

Post-fabrication Engineering of the Membranes

For further improvement of the aspect ratio, the present inventors alsosuggest to etch the polymeric membrane by using dry plasma orwet-etching in order to reduce membrane thickness, while keeping theaperture diameter almost constant (FIG. 5). This in turn enhances theaspect ratio. The present inventors also expect that the edges of theaperture may lose their initial rectangular cross-section shape and mayadapt a more triangular cross-section, which leads to a conic shape ofthe aperture.

In the case of extended pores as described above, dry etching orwet-etching could be used to not only thin the “layer 1” in a controlledfashion, but also to partially remove “layer 1” from the edge of theunderlying “layer 0” (FIG. 6). The resulting edge structure consists of“layer 2” as the major supporting layer, “layer 1” as another supportinglayer and the edge ring of “layer 0”, which could be very thin on theone hand (larger aspect ratios) and subject to tailored surfacemodification on the other hand.

Surface Modification:

The present inventors also suggest a number of surface modifications ofthe membranes—mainly in order to enhance the hydrophobicity of themembrane surface. The purpose of the hydrophobic surface is to improvethe wetting of an organic phase (so-called “pre-treatment”) that formsthe annulus (gasket) that supports the bilayer. The inventors expectthat the enhanced membrane wetability with the organic phase enhancesthe formation and the stability of bilayers across the aperture.

The modification may include a surface activation, cleaning,roughness-enhancement step (e.g., by Ar, CHF₃, CF₄, O₂ plasma andcombinations), which may be followed by the deposition of functionallayers through dip-coating, gas-phase deposition, or evaporationmethods. Preferred functional layers consist of one of the followingmaterials: fluorosilanes, alkysilanes, fluorinated components from therespective plasma used.

Moreover, reference is made to the figures, wherein

FIG. 1 shows the basic principle of the design of a polymeric membranehaving a tapered pore and having a lipid bilayer and a pore proteininserted therein, in accordance with the present invention.

FIG. 2 shows a schematic view of the tapered pore formed by thetwo-layered/multilayered structure of the membrane.

FIG. 3 shows the manufacturing of such tapered pore.

FIG. 4 shows a variation of the process shown in FIG. 3.

FIG. 5 shows yet another variation of the manufacturing process.

FIG. 6 shows yet a further variation of the manufacturing process.

FIG. 7 a shows an SEM image of a resulting membrane/aperture structure.The central aperture diameter is 20 μm. The height of the SU8 layer is500 nm.

FIG. 7 b shows an SEM image of a central 4.5 μm wide aperture in a 1.5μm thick SU8 layer.

FIG. 8 shows an SEM image of the back of an example membrane inaccordance with the present invention.

FIG. 9 shows the relationship between oxygen plasma etching time and theresulting thickness of a membrane.

FIGS. 10 a and 10 b show the SEM images of an oxygen plasma etched SU8membrane.

FIGS. 11 a and 11 b show the results of SEM morphology measurements andwater contact angle and AFM roughness analysis.

FIGS. 12 a and 12 b show SEM morphology measurements and water contactangle and AFM roughness analysis.

FIG. 13 shows an example of a 3×3 array of tapered pores in SU8 resist.

FIG. 14 shows a biosensor performance chart using membranes inaccordance with the present invention in comparison with laser-drilledmembranes.

FIG. 15 shows the biosensor performance score chart for surface modifiedSU8 membranes in accordance with the present invention.

Moreover, reference is made to the following examples, which are givento illustrate, not to limit the present invention.

EXAMPLE 1 Tapered Pore Fabrication

The steps of a fabrication approach that realises a 10 μm wide aperturein a 500 nm thick polymeric membrane are shown in FIG. 3. A siliconwafer with native silicon oxide on top (Cyrstec GmbH, Berlin, Germany)is covered with a 40 nm thick, thermally evaporated anti-sticking layer,e.g. a 40 nm thick, thermally evaporated layer of gold (FIG. 3,a)(=layer 0). Then, the first membrane layer is spin-coated onto the gold(FIG. 3,b) (=layer 1). In this example, the present inventors usednegative-tone SU8 photoresist (Micro Resist Technology GmbH, Berlin,Germany) of the so-called 2000.5 formulation (Cat.-No.:SU8 2000.5, MicroResist Technology GmbH, Berlin, Germany), which gives a 500 nm thicklayer is spin-coated at 3000 rpm for. After soft-baking at 65° C. for 1min and 95° C. for 2 minutes, the resist layer was exposed at 365 nm to400 nm wavelength using a MJB3 contact aligner (Karl Suss GmbH, Germany)using a dose of about 250 mJ/cm2. The applied mask was specificallydesigned in order to pattern a circular, rectangular, or squaredmembrane with an area in the range of several mm² and an aperture/porein its centre. The post-exposure bake step consists of a bake at 65° C.for 1 minute and at 95° C. for 2 minutes.

After developing the layer for 2 minutes using SU8-developer (MicroResist Technology GmbH, Berlin, Germany) (FIG. 3,c), a second SU8 resistlayer (“supporting layer”) (=layer 2) was spun onto the first layer(FIG. 3,d). In this case the SU8 2007 formulation was selected, whichresults in a about 13 μm thick layer, if spun at 1000 rpm. After asoft-bake at 65° C. for 2 minutes and at 95° C. for 3 minutes, thesupporting layer was exposed using the same aligner and a UV light doseof about 350 mJ/cm². During this step, the wider aperture in thesupporting layer was carefully aligned onto the smaller aperture in thelayer 1 by using alignment marks. After the post-exposure bake at 65° C.for 1 minute and at 95° C. for 2 minutes, the structure was developedfor 2.5 minutes. (FIG. 3,e)

Peeling step: after these steps, the membrane could be lifted bygrapping it with a sharp tweezer or any other suitable tool. Themembrane easily releases from the substrate due to the week adhesionbetween the gold-layer and the siliconoxide substrate. (FIG. 3,f)

Gold-removal step: The gold layer is then removed either by dryargon-etching or by wet-chemical etching, e.g., KI/I₂ etching. FIGS. 7 aand 7 b show an SEM image of the resulting membrane/aperture structure.

EXAMPLE 2 Tapered Pore with Gold Backside-layer

This example describes the realisation of a tapered pore, which isextended through a gold-backside layer that is used as an anchor layerfor functional molecules.

The process is performed as described in Example 1. The peeling step isnot performed at this stage, but instead of this, an Ar-plasma etchingstep or any other suitable etching step is inserted, during which thegold is completely removed from the aperture region. (FIG. 4 a-d).

After this, the membrane layer can be lifted by peeling as described inExample 1. FIG. 8 shows SEM images of the back of an example membrane.

EXAMPLE 3 Post-fabrication Engineering: Tapered Pores with ThinnedMembrane for very Large Aspect Ratios

As described above, the aspect ratio of the fabricated apertures in thepolymeric membranes could be improved by reducing the membrane thicknessby dry or wet etching. Here the inventors present an example of usingoxygen plasma etching. An SU8 membrane system was fabricated asdescribed in Example 1. Then, as shown in FIG. 5 a-d, the wafer wasintroduced into a reactive ion etching machine (PlasmaLab 80, OxfordInstruments, Germany) and oxygen plasma was applied at 200W fordifferent etching times. FIG. 9 shows the relationship between the O₂plasma etching time and the resulting thickness of an initially 500 nmthick SU8 membrane. FIGS. 10 a and 10 b show the SEM images of an O₂plasma etched SU8 membrane. The thickness of the central membrane isabout 150 nm, the aperture diameter is 20 μm, resulting in an aspectratio of about 130:1.

In a further variation, the aperture is first etched through thegold-back layer (see Example 2) and then an oxygen-plasma is used tothin the “layer 1” (FIG. 3 b,c) and—at the same time to slightlyincrease the diameter of the aperture in “layer 1” as shown in FIG. 6 c.

EXAMPLE 4 Surface Modified Membranes EXAMPLE 4.1 Silanisation in LiquidPhase

Prior to the silanisation in toluene, the membrane surface was cleanedand covered with OH-groups by applying a short oxygen plasma treatment(PlasmaLab 80, Oxford Instruments, Germany). 1-10 seconds plasma time at100-200 W plasma power and 50-100 mbar background pressure are usuallysufficient to activate the SU8 surface. The silane component was dilutedin water-free toluene to achieve a volume concentration of about 1-3%.The activated SU8 surface was immersed into the silane solution for10-60 minutes, washed with fresh toluene and dried.

EXAMPLE 4.2 Silane Vapour Deposition

For vapour deposition of silane, 100-500 μl of the pure silane solutionwas either heated in a oil bath to temperatures between 100° C. and 160°C. (depending on the type of silane, mainly in case of ethoxy-silanes)or just brought into a vacuum chamber (e.g. in case of chloro-silanes).The background pressure was adjusted to about 0.5 mbar after insertionof the silane. The activated SU8 surface was brought either into thevapour of the heated silane or into the vacuum chamber and remainedthere for 20-60 minutes. Usually, contact angle and XPS measurementsindicated that silanes were successfully bound to the SU8 surface.

Example 4.3 Surface Modification by Plasma Treatment

A reactive ion etching machine (PlasmaLab 80, Oxford Instruments,Germany) was used to modify the surface of for example SU8 membranes.Ar, O₂, CHF₃, CF₄ plasmas and mixtures hereof have two effects on themembrane surface. Firstly, they damage the surface by partial removal ofmaterial. This effect usually increases the surface roughness of thetreated surface. Secondly, CHF₃ and CF₄ plasmas are known to partiallycover the treated surfaces with fluorinated components. By using thereactive ion etching plasma approach, the present inventors couldcombine both effects and found that the treated surfaces show very highcontact angles of more than 100°.

CHF3/Ar Plasma Treatment

SU8 membranes such as fabricated in Example 1 were inserted into thePlasmaLab 80 machine and treated for 1 to 10 minutes at a CHF3 flow of25 sccm and an Ar flow of 25 sccm. The background pressure was 30 mT andthe plasma generator power was 200 W. The results of SEM morphologymeasurements, as well as water contact angle and AFM roughness analysisare shown in FIGS. 11 a and 11 b, respectively.

CHF3/CF4 Plasma Treatment

SU8 membranes such as fabricated in Example 1 were inserted into thePlasmaLab 80 machine and treated for 1 to 10 minutes at a CHF3 flow of20 sccm and an CF4 flow of 20 sccm. The background pressure was 25 mTand the plasma generator power was 300 W. The results of SEM morphologymeasurements, as well as water contact angle and AFM roughness analysisare shown in FIGS. 12 a and 12 b, respectively.

For both types of plasmas, a strong increase of the contact angle can beseen already after a few minutes of treatment. At the same time, theroughness of the surfaces increases with plasma treatment time. Theroughness of the CHF3/CF4 plasma treated surface is slightly smaller incomparison to the CHF3/Ar plasma treated surface.

EXAMPLE 5 Arrays of Tapered Pores

In order to satisfy the requirement of quick protein insertion also forthe case of very small-area bilayers, the invented aperture structurescan be arrayed to provide an effectively larger bilayer surface, whilekeeping the area of each individual bilayer advantageously small. FIG.13 shows an example of a 3×3 array of tapered aperture in SU8 resist.

EXAMPLE 6 Bilayer Formation & Statistics in Comparison to ClassicalApertures

FIG. 14 shows a biosensor performance chart for SU-8, 10 μm taperedapertures versus 10 μm laser drilled membranes using standardpretreatment conditions. The term “zappable” as used herein inconnection with a bilayer means that the bilayer can be destroyed by ashort voltage pulse of several hundred of mV. The pore protein that wasinserted in these experiments was alpha-hemolysine. Results shows that10 μm tapered membranes perform better at forming bilayers with largeareas for easy pore protein insertion (Biosensor score 8). The evidenceshown here is strong.

The “Biosensor Scoring” scheme is explained in the following:

-   -   10—Controlled number of pore proteins inserted    -   9—Bilayer formed first time, pore proteins inserted first time    -   8—Bilayer formed, pore proteins inserting    -   7—Initially blocked, but bilayer eventually formed with pore        proteins inserting    -   6—Bilayer formed, pores insert, but bilayer lifetime is short    -   5—Zappable bilayer formed but no pore proteins insert    -   4—Initially blocked, but eventually zappable bilayer formed (no        pore proteins)    -   3—Bilayer forms relatively easily (zappable), but has short        lifetime    -   2—Unzappable blockage which can't be removed    -   1—Unbreakable connection after solution applied    -   0—Not scored

FIG. 15 shows the biosensor performance score chart for surface modifiedSony SU-8 membranes, i.e. examples membranes formed in accordance withthe present invention. These results suggest that the chances of firstpass bilayers with pores (Biosensor score 9) are improved byfluorosilane.

The membranes in accordance with the present invention are cheaper incomparison to silicon-based membranes, they provide very high aspectratios at low aperture diameters around 10μm. Moreover, the membranes inaccordance with the present invention are very thin and yet stable andcan therefore be handled easily with a reduced risk of rapture.

REFERENCES

1 WO9425862A1, Dogan et al., “Biosensor substrate for mounting bilayerlipid membrane containing a receptor”.

2 EP1712909 (A1), US2007161101 (A1), WO2005071405, S. Takeuchi et al.,“Method of forming planar lipid double membrane for membrane proteinanalysis and apparatus therefore.”

3 US2006228402, EP1710578 (A1), Pohl et al. “Techniques for forming alipid bilayer membrane”

4 US2004120854A1, R. Pantoja, J. Heath, et al., “Silicon-wafer baseddevices and methods for analyzing biological materials”; R. Pantoja, J.Heath, et al. Biophys. J, 2001, 81, 2389-2394.

5 Olgier et al. Langmuir, “Suspended Planar Phospholipid Bilayers onMicromachined Supports” 16, 2000 5696-5701.

6 Olgier et al. Langmuir, “Single Ion Channel Sensitivity in SuspendedBilayers on Micromachined Supports” 17, 2001 1240-1242.

7 WO2006119915A1, EP1721657A1, Harnack et al., “A method of fabricatinga polymeric membrane having at least one pore”.

8 Mayer et al., Biophys. J “Microfabricated Teflon Membranes forLow-Noise Recordings of Ion Channels in PlanarLipid Bilayers”, 2003,85(4) 2684-2695.

9 White et al., Biophys. J. “Analysis of the Torus Surrounding PlanarLipid Bilayer Membranes”, 1972, 12,432-445.

The features of the present invention disclosed in the specification,the claims and/or in the accompanying drawings, may, both separately andin any combination thereof, be material for realizing the invention invarious forms thereof.

1. A method of fabricating a membrane having a tapered pore, said methodcomprising the steps: a) providing a substrate, b) depositing ananti-sticking layer on said substrate, c) depositing a first polymericlayer of a first polymeric material on said anti-sticking layer, d)introducing a first aperture into said first polymeric layer, said firstaperture having a first diameter, e) depositing a second polymeric layerof a second polymeric material onto said first polymeric layer, whichsecond polymeric layer is located on said first polymeric layer andwherein a part of said second polymeric material also extends into andfills said first aperture, f) introducing a second aperture into saidsecond polymeric layer, said second aperture having a second diameterwhich is larger than said first diameter, said second aperture beingaligned with said first aperture, such that said part of said secondpolymeric material, which extends into and fills said first aperture,becomes removed, g) repeating steps e) and f) n-times, thus generating athird, fourth, . . . (n+2)th polymeric layer having a third, fourth, . .. (n+2)th aperture in said third, fourth, . . . (n+2)th polymeric layer,said third, fourth, . . . (n+2)th aperture having a third, fourth, . . .(n+2)th diameter which is increasingly larger with increasing n, n beingan integer from 0 to 10, said method thus resulting in a polymericmembrane on an anti-sticking layer and a substrate, said polymericmembrane having a tapered pore comprising (n+2) apertures of increasingdiameter, said (n+2) apertures being aligned with each other to formsaid tapered pore.
 2. The method according to claim 1, wherein said(n+2) apertures are concentrically aligned.
 3. The method according toany of the foregoing claims, wherein said first diameter is in the rangefrom 1 nm to 100 μm, preferably from 1 nm to 10 μm.
 4. The methodaccording to any of the foregoing claims, wherein said first polymericlayer has a height in the range from 0.3 nm to 20 μm, preferably 0.3 nmto 2 μm, and wherein said second and, if present, third, fourth, . . .(n+2)th polymeric layer has a height in the range from 500 nm to 1 mm,preferably 5 μm to 50 μm.
 5. The method according to any of theforegoing claims, wherein the ratio of said first diameter: height ofsaid first polymeric layer is ≧3.
 6. The method according to any of theforegoing claims, wherein said substrate is made of a material selectedfrom the group comprising glass, silicon, silicon oxide, siliconnitride, GaAs, saphire, polycarbide, polycarbonate.
 7. The methodaccording to any of the foregoing claims, wherein said anti-stickinglayer is made of a material selected from the group comprising metalswith a weak adhesion to the substrate, such as gold, silver, platinum,titanium, aluminum, alloys hereof, fluorosilanes, mica, carbon,water-soluble materials (e.g. CaO, Ca(OH)₂), heat-disposable materialsand has a thickness in the range from 10 nm to 100 nm.
 8. The methodaccording to any of the foregoing claims, wherein said first, secondand, if present, third, fourth, . . . (n+2)th polymeric material is,independently, at each occurrence, selected from the group comprising ofresists for optical lithography, electron beam lithography, and imprintlithography.
 9. The method according to any of the foregoing claims,wherein step b) occurs by a method selected from evaporation,sputtering, e-gun evaporation, gas-phase-deposition, sublimation,electro-chemical deposition.
 10. The method according to any of theforegoing claims, wherein steps c) and e) are independently performed bya method selected from spin coating, dip coating, spray coating, vacuumdeposition, Langmuir Blodgett techniques, deposition from the gas-phase.11. The method according to any of the foregoing claims, wherein stepsd) and f) are independently performed by a method selected from opticallithography, electron beam lithography, imprint lithography, and focusedion beam etching.
 12. The method according to any of the foregoingclaims, wherein in steps d) and f), a plurality of first and secondapertures are introduced.
 13. The method according to any of claims8-12, wherein said resists for optical lithography, electron beamlithography and imprint lithography are selected from the groupcomprising negative and positive tone resists for optical lithography,electron beam lithography and imprint lithography.
 14. The methodaccording to any of the foregoing claims, further comprising the step:h) removing said substrate by peeling off said polymeric membrane on andtogether with said anti-sticking layer from said substrate.
 15. Themethod according to claim 14, further comprising the step: i) removingsaid anti-sticking layer by dry etching, such as dry argon-etching, O₂plasma etching or wet chemical etching, such as KI/I₂-etching to removegold, etching using a strong base to remove aluminum.
 16. The methodaccording to any of claims 1-13 or 14, further comprising the step: i′)introducing an aperture into said anti-sticking layer by dry etching,such as dry argon-etching, or wet chemical etching, such asKI/I₂-etching, and using said first polymeric layer as an etching mask.17. The method according to any of claims 1-13, further comprising thestep: h′) selectively etching said polymeric membrane on saidanti-sticking layer and said substrate by using dry plasma etching, suchas O₂ plasma etching, or chemical wet etching, such as oxidation inH₂SO₄/H₂O₂, to reduce the total thickness of said polymeric membrane,and, optionally, removing said substrate by peeling off said polymericmembrane on and together with said anti-sticking layer from saidsubstrate.
 18. The method according to claim 16, further comprising thestep: k) selectively etching said polymeric membrane on saidanti-sticking layer and, if present, on said substrate, by using dryplasma etching, such as O₂-plasma etching, or chemical wet etching, suchas oxidation in H₂SO₄/H₂O₂ to reduce the total thickness of saidpolymeric membrane, and, optionally, removing said substrate, ifpresent, by peeling off said polymeric membrane on and together withsaid anti-sticking layer from said substrate.
 19. The method accordingto any of claims 14-18, wherein, after removal of said substrate, saidpolymeric membrane and/or, if present, said anti-sticking layer is/arefurther surface modified by a method selected from a) immobilizingfunctional groups or molecules thereon to match the surface propertiesof a lipid bilayer to be introduced into said first aperture or intosaid aperture in said anti-sticking layer, b) surface activation byintroducing OH-groups through O₂-plasma treatment c) enhancing thesurface roughness by etching, such as Ar, CHF₃, CF₄, O₂-plasma andcombinations thereof, d) deposition of one or several functional layers,e.g. by dip coating, gas-phase deposition or evaporation, and anycombination of steps a)-d).
 20. The method according to claim 19,wherein said functional layer is selected from fluorosilanes,alkylsilanes, fluorinated plasma components from CHF₃, CF₄.
 21. Apolymeric membrane having a tapered pore and comprising (n+2) polymericlayers as defined in any of claims 1-20, wherein said tapered pore isformed by (n+2) apertures of differing diameter within said (n+2)polymeric layers, n being an integer from 0 to 10, said n+2 aperturesbeing aligned with each other to form said tapered pore.
 22. Thepolymeric membrane according to claim 21, wherein said (n+2) aperturesare concentrically aligned.
 23. The polymeric membrane according to anyof claims 21-22, wherein said tapered pore, at its smallest diameter,has a ratio of diameter of said pore to height of said polymeric layerat said smallest diameter of said pore of ≧3.
 24. The polymeric membraneaccording to any of claims 21-23, fabricated by the method according toany of claims 1-20.
 25. The polymeric membrane according to any ofclaims 21-24, having a plurality of tapered pores, as defined in any ofclaims 21-24.
 26. The polymeric membrane according to any of claims21-25, further comprising a lipid bilayer spanning the pore at itssmallest diameter and, optionally, having biological membrane proteins,such as pore proteins or channel proteins incorporated in said lipidbilayer.
 27. Use of a membrane according to any of claims 21-25 forforming a lipid bilayer.
 28. Use of a polymeric membrane according toclaim 26 for physiological measurements, such as patch clampmeasurements or as an electronic sensor.